Preoperatively predicting the lymph node metastasis and prognosis for gastric cancer patients

The preoperative distinguishment of lymph nodes (LN) with metastasis plays a pivotal role in guiding the surgical extension for gastric cancer (GC). We aim to identify the preparative risk factors for LN metastasis in GC patients. We retrospectively reviewed 424 patients who underwent radical GC resection in our medical center between Jan 2011 and Dec 2018. Multivariate logistic regression was employed to identify risk factors for LN metastasis, while multivariate COX regression was utilized to evaluate prognostic factors. The median overall survival of patients with or without LN metastases was 31 and 58 months, respectively. In multivariate analysis, lower albumin (OR = 0.512; P = 0.004) and prealbumin (OR = 0.367, P = 0.001) and higher CEA (OR = 3.178, P < 0.001), CA199 (OR = 2.278, P = 0.002) and platelets (OR = 1.697, P = 0.017) were found to be significantly associated with LN metastasis. In survival analysis, older age (HR = 1.712), larger tumors (HR = 1.082), higher D-dimer (HR = 1.561) and CA199 (HR = 1.553), advanced staging (stage II, HR = 3.446; stage III-IV, HR = 11.089), lower prealbumin levels (lower level for reference, HR = 0.63), and absence of adjuvant chemotherapy (HR = 0.396) was discovered to be associated with poorer overall survival (all P < 0.05). In conclusion, our results demonstrated that preoperative prealbumin-bound tumor markers can effectively predict LN metastasis. Additionally, prealbumin was found to possess prognostic value as well.


Risk factors for LN metastasis
The cutoff values for CEA, CA199, albumin, prealbumin, FIB, D-dimer, FDP, hemoglobin, platelets, leukocytes, lymphocytes, monocytes, and neutrophils were 4.42 ng/mL, 19.07 U/mL, 39.75 g/L, 159.1 mg/L, 3.78 g/L, Table 1.Demographic and baseline characteristics of patients grouped by lymph node (LN) status.Values in parentheses are percent or interquartile ranges.FDP fibrin degradation products, CEA carcinoembryonic antigen, CA199 carbohydrate antigen 199, WBC white blood cell, Hb hemoglobin, OS overall survival, DFS disease free survival.Univariate logistic regression analysis showed that lower levels of albumin, prealbumin, and hemoglobin, as well as higher levels of FIB, D-dimer, CEA, CA199, white blood cell (WBC) count, neutrophils, monocytes, and platelets, were identified as risk factors for LN metastasis.
To evaluate lymph node metastasis more efficiently, we conducted a combined ROC curve analysis.The analysis, which combined CEA and CA199 (referred to as Combined P1), yielded an area under the curve (AUC) of 0.664, a sensitivity of 0.531, and a specificity of 0.765.Additionally, the ROC analysis that combined CEA, CA199, albumin, prealbumin, and platelets (referred to as Combined P2), indicated an AUC of 0.736, a sensitivity of 0.59, and a specificity of 0.803 (Fig. 1a).Importantly, according to Table 4, the diagnostic efficacy of Combined P2 was significantly higher than that of Combined P1 (P < 0.001).
Table 2. Univariate and multivariate analyses of variables for lymph node metastasis.The optimal cut-off value was 39.75 g/L for albumin, 159.1 mg/L for prealbumin, 3.78 g/L for fibrinogen, 0.965 mg/L for D-dimer, 0.65 mg/L for FDP, 4.42 ng/mL for CEA, 19.07 U/mL for CA199, 4.915 *10^9/L for WBC, 113.5 *10^9/L for Hb, 3.12 *10^9/L for neutrophil, 1.745 *10^9/L for lymphocyte, 0.366 *10^9/L for monocyte and 188.5 *10^9/L for platelet.The reference for gender, age and peripheral blood indexes were female, age < 60 years and the lower level of peripheral blood indexes according to their cutoff values.CI confidence interval.
Table 5. Univariate and multivariate analyses for overall survival of GC patients.The cut-off value was 39.75 g/L for albumin, 159.1 mg/L for prealbumin, 3.78 g/L for fibrinogen, 0.965 mg/L for D-dimer, 0.65 mg/L for FDP, 4.42 ng/mL for CEA, 19.07 U/mL for CA199, 4.915 *10^9/L for WBC, 113.5 *10^9/L for Hb, 3.12 *10^9/L for neutrophil, 1.745 *10^9/L for lymphocyte, 0.366 *10^9/L for monocyte and 188.5 *10^9/L for platelet.The reference of gender, age, adjuvant chemotherapy, differentiation and cancer embolus were female, < 60 years, without chemotherapy, poorly differentiation and no cancer embolus.All peripheral blood indexes were divided into two groups according to the cutoff value, and low levels were used as the control group for survival analysis.HR hazard ratio, CI confidence interval.

Discussion
Our current study demonstrates the significance of albumin, prealbumin and platelets in preoperative peripheral blood for LN metastasis in GC.The ROC curve indicated that the utility of albumin, prealbumin and platelets as predictive markers for LN metastasis in GC was comparable to that of traditional GC markers, namely CEA and CA199.The combined application of these indicators improves the efficacy for predicting LN metastasis.Prealbumin was also an independent factor for patient survival in all analyzed blood parameters, as were as D-dimer and GC markers.Neoadjuvant chemotherapy has shown a survival benefit relative to surgery alone 8 , and the possibility of LN metastasis is a key consideration for such treatment.Neoadjuvant chemotherapy is recommended for patients with lymph node metastases in Japan 9 .It has been reported that neoadjuvant chemotherapy can effectively control LN metastasis in GC, thereby reducing tumor N stage and increasing complete resection 10 .Therefore, preoperative assessment of LN status provides a reference for choosing a reasonable treatment plan for patients.
Tumor markers are commonly used to predict prognosis, monitor a positive course, and detect recurrence 11,12 .As traditional tumor markers, CEA and CA199 may help detect positive lymph nodes 13,14 .In our current study, we assessed the predictive value of preoperative peripheral blood-derived clinical routine laboratory biomarkers for LN metastasis.Our data showed that the LN metastasis group had significantly elevated CEA and CA199.Multivariate regression analysis showed their clinical significance for predicting LN metastasis.Previous studies reported that CEA, CA199, CA724 and CA242 were all associated with LN metastasis, and their combination could improve diagnostic efficacy 15 .This is consistent with our conclusions, although we only analyzed CEA and CA199 without consideration of CA724 and other GC markers.
In addition to the aforementioned tumor markers, we also found that platelets and nutritional markers (albumin and prealbumin) may independently predict LN involvement.In fact, studies have reported that platelet Table 6.Univariate and multivariate analyses for progression-free survival of GC patients.HR, hazard ratio; CI, confidence interval.The cut-off value was 39.75 g/L for albumin, 159.1 mg/L for prealbumin, 3.78 g/L for fibrinogen, 0.965 mg/L for D-dimer, 0.65 mg/L for FDP, 4.42 ng/mL for CEA, 19.07 U/mL for CA199, 4.915 *10^9/L for WBC, 113.5 *10^9/L for Hb, 3.12 *10^9/L for neutrophil, 1.745 *10^9/L for lymphocyte, 0.366 *10^9/L for monocyte and 188.5 *10^9/L for platelet.The reference of gender, age, adjuvant chemotherapy, differentiation and cancer embolus were female, < 60 years, without chemotherapy, poorly differentiation and no cancer embolus.All peripheral blood indexes were divided into two groups according to the cutoff value, and low levels were used as the control group for survival analysis.www.nature.com/scientificreports/count may be a reliable biomarker of lymph node metastasis likelihood 16,17 .Not only that, platelet-derived markers such as platelet-lymphocyte ratio are also useful biomarkers for predicting LN metastasis 18 .Platelets promote cell proliferation, angiogenesis, and epithelial-mesenchymal transition, and protect tumor cells from immune system attack by interacting with tumor cells to form microthrombi 19,20 .Therefore, the correlation between platelets and LN metastasis is reasonable.Accumulating evidence suggests that tumor-associated inflammation is tumor-promoting 21 .In our study, we mainly focused on the role of a single blood marker and therefore did not consider various derived markers of systemic inflammation.Although we did not find an independent predictive role of inflammatory cells and lymphocytes on LN metastasis, platelets showed an advantage.By interacting with inflammatory cells and lymphocytes, platelets are important coordinators of inflammation as well as immune responses 22 .Prealbumin, also known as transthyretin, has a shorter half-life than albumin, only 2-3 days, and its levels are mainly affected by liver function and inflammation 23 .Notably, we found that both albumin and prealbumin were independent predictors of LN metastasis.It has been identified that perioperative prealbumin might be useful in predicting postoperative early recurrence of lung cancer and short-term postoperative outcomes after gastrectomy 24,25 .Although there are few studies on the assessment of LN metastasis by albumin or prealbumin, the combination of prealbumin and inflammatory response index has been implicated in metastasis in GC patients 26 .A study demonstrates the predictive value of platelet-to-albumin ratio for LN metastasis 5 .Given that both platelets and albumin are independent predictors of LN metastasis, our results are consistent with this report.In our study, we did not analyze the clinical significance of the variables calculated from peripheral blood parameters, and it is not difficult to infer that the ratio of platelets to albumin or the ratio of platelets to prealbumin would have a higher predictive value for LN metastasis.In order to more effectively assess the status of LN metastasis, we performed a combined ROC analysis and found that combining CEA, CA199, platelets, albumin, and prealbumin significantly improved the predictive power for LN metastasis compared to combining CEA and CA199 alone.This inspires clinicians that low prealbumin levels before treatment can not only guide nutritional support, but also reflect the status of LN metastasis.The risk of LN metastasis can be further evaluated according to the preoperative blood index in patients with N0 who have not been proved by imaging methods.
GC patients are often accompanied by elevated coagulation markers, such as fibrinogen and D-dimer.In fact, crosstalk between coagulation and inflammation makes them mutually reinforcing and is closely related to tumor progression and prognosis 27 .Studies have shown that preoperative plasma fibrinogen is associated with LN metastasis.Although computed tomography (CT) was more valuable in predicting lymph node metastases, CT could not predict lymph node metastases in 20.6% of patients, and 53.8% of them had plasma fibrinogen above the cutoff value 28 .The predictive value of D-dimer for LN metastasis has also been reported 29 .However, multivariate logistic regression showed that coagulation related markers were not independent risk factors for LN metastasis.Our current study did not compare the predictive power between blood parameters and CT, but the combination of CT and pre-treatment blood parameters can provide evidence for evaluating LN metastasis and then formulating treatment strategies.
Nutrition-related indicators are most used to guide perioperative nutritional support and evaluate the prognosis of tumor patients 30,31 .Prealbumin is more sensitive to changes in protein-energy status than albumin, therefore more suitable for nutritional monitoring 32 .In terms of survival of patients, albumin and prealbumin are well known as prognostic factors.But few studies compare prognostic superiority of albumin and prealbumin.After our analysis, we found that prealbumin, but not albumin, was an independent risk factor for OS and DFS in GC.Among other preoperative blood parameters, tumor markers (CA199 for OS and CEA for DFS) and D-dimer were independently associated with gastric cancer prognosis, which is consistent with previous studies.
This study has some limitations.First, it is a retrospective study with a small sample size.Second, we only focus on a subset of peripheral blood indicators before treatment, such as inflammatory status through blood cells.However, other indicators of this status, such as C-reactive protein, were not included in the analysis.Similarly, among gastric cancer markers, we only analyzed CEA and CA199.Third, we only considered the individual effects of each index, and did not analyze other markers calculated by these factors, such as NLR, SII, etc.However, we performed a joint ROC analysis by multivariate logistic regression.Fourth, as an effective method for detecting lymph node metastasis, imaging methods have not been compared with those of peripheral blood in their diagnostic performance.

Conclusions
In conclusion, preoperative prealbumin-bound tumor markers can effectively predict LN metastasis in GC.Additionally, prealbumin was found to possess prognostic value as well.

Methods
We retrospectively enrolled 424 patients with GC who underwent radical resection in the Department of Oncology, First Affiliated Hospital of Xi'an Jiaotong University from January 2011 to December 2018.All patients had undergone their initial diagnosis and had not received preoperative chemoradiotherapy.Patients were followed up by telephone, and the last follow-up was in December 2020.All patients underwent radical surgery (open or laparoscopic), and pathological staging was assessed according to the eighth edition of the American Joint Cancer Society (AJCC) tumor-node-metastases (TNM) classification.The involvement of LN was confirmed through a postoperative pathological examination.The patient's overall/disease-free survival was defined as the time from the date of surgery to the patient's death/relapse or last follow-up.Pretreatment laboratory measurements include whole blood cells, tumor markers (CEA, CA199), coagulation markers (fibrinogen, D-dimer and fibrin degradation products [FDP]), albumin and prealbumin.This study was performed in line with the principles of the Declaration of Helsinki.Approval was granted by the Ethics Committee of First Affiliated Hospital of Xi'an

Figure 1 .
Figure 1.ROC curves and Kaplan-Meier survival curves.(a) ROC curve analysis for predicting lymph node metastasis.Pre-markers only represent ROC curve combining CEA and CA199; pre-combined represent ROC curve combining CEA, CA199, albumin, prealbumin and platelet.(b) Kaplan-Meier survival curves of prealbumin for overall survival (OS); the 5 year survival rate of low prealbumin group was 29.8%, of high prealbumin group was 62.6%.(c) Kaplan-Meier survival curves of prealbumin for disease-free survival (DFS); the 5 year survival rate of low prealbumin group was 28.6%, of high prealbumin group was 60.1%.

Table 3 .
Multivariate multiple logistic regression analyses of variables for LN metastasis.For multiple logistic regression analyses the reference category is N0 stage.The reference of parameters was albumin ≥ 39.75, prealbumin ≥ 159.1, CEA ≥ 4.42, CA199 ≥ 19.07 and platelet ≥ 188.5.